Miniaturization of analytical methods and instrumentation for biomedical and clinical research is an area of burgeoning interest. In many cases, the reduction in size of an analytical procedure or technique often translates to a reduction in analysis time and costs. Miniaturization of analytical methods often paves the way for the use of established technologies in high-throughput applications. Great efforts have been made to develop fast, cost-effective, high-throughput separation methods for nucleic acid analysis. For example, microchip technology is currently being developed in which rapid thermocycling and electrophoretic separation can be accomplished 10 times quicker than conventional techniques. The microchip platform has the potential for integrating sample pretreatment, target amplification, and detection in a single device. Combination of these processes into a single device (i.e., create the elusive “lab-on-a-chip”) can minimize sample loss and contamination problems as well as reduce analysis times substantially.
Purification of nucleic acids from biological sources, while commonplace, is not a trivial challenge. For example, one of the simplest sources of human genomic DNA is white blood cells (WBCs). One microliter of whole blood contains ˜5000 WBCs which, in turn, contain a total of ˜35 ng of DNA. While effective PCR for molecular biological analysis requires only a few copies of the genome, the more efficient the recovery of DNA, the more effective the PCR amplification. Efficient capture and purification of DNA can be affected by the presence of several PCR inhibitors in whole blood (e.g., heme from hemoglobin) as well as the numerous other components present including lipids, proteins, small ions and peptides. For this reason, purification of DNA from such systems is nontrivial.
While the functional integration of sample preparation, DNA amplification, and sample analysis in a single electrophoretic microchip has obvious advantages, the potential of a miniaturized DNA purification method extends beyond reducing analysis time in molecular diagnostics. A miniaturized DNA purification method would have further utility in a number of other areas including separating PCR product from reaction by-products, purifying DNA fragments prior to sequencing, and desalting primers of DNA hybridization targets. An effective miniaturized DNA sample preparation methodology could also be interfaced with conventional capillary electrophoresis, integrated electrophoretic microchips or pipettes capable of micro-solid-phase extraction (μSPE). An optimal miniaturized DNA purification protocol will accomplish extraction and purification of DNA in as few steps as possible and should minimize solvent volume, lower dilution effects, and reduce the possibility of contamination.
Although methods that exploit the proclivity of certain materials for adsorbing DNA (e.g., silica, glass fibers, anion exchange resins and modified magnetic beads) have been developed for purifying DNA, little information is available regarding the design and operation of a miniaturized DNA purification method based on silica resins. Specific problems associated with μSPE devices include the total capacity of the μSPE device, the compatibility of the retained DNA fraction with PCR applications, and the reproducibility of the DNA extraction method with complex clinical samples.